大黄素抑制血管平滑肌细胞增殖机制研究

【作者】 王翔飞；

【导师】 葛均波；

【作者基本信息】 复旦大学 ， 内科学， 2005， 博士

【摘要】 第一部分大黄素抑制血管平滑肌细胞增殖机制研究背景：大黄素(1，3，8-三羟基-6-甲基葸醌)是天然蒽醌化合物，可以抑制多种肿瘤细胞增生。然而，大黄素对平滑肌细胞的作用机制研究甚少。大黄素能够促进肿瘤细胞(Mahlavu，HL-60)凋亡，Mahlavu细胞凋亡现象可被抗氧化剂(过氧化氢酶和环孢霉素A)部分阻断，但是HL-60细胞凋亡现象不能被抗氧化剂所阻断，因此ROS途径是否参与大黄素诱导细胞凋亡存在争议。大黄素在细菌中常有致突变效应(mutagenicity)，在细胞中常有基因毒性(genotoxicity)。DNA损伤促进p53表达，p53途径活化后既可以抑制细胞生长，又可促进细胞凋亡。ROS途径和p53途径是大黄素发挥作用的可能途径。大黄素可被细胞色素P450代谢酶(CYP)代谢转化为2-羟基大黄素，它抑制增殖效果强于大黄素。CYP抑制剂或者诱导剂可能改变大黄素作用特点。方法：人血管平滑肌细胞(VSMCs)培养于M1 99培养液中。通过细胞计数、MTT法、细胞周期、细胞迁移等方法观察大黄素的作用特点。使用Annexin V和7-AAD双染法、乳酸脱氢酶释放来观察大黄素促VSMCs死亡的方式。对于ROS途径，利用ROS生成阻断剂观察大黄素促ROS生成途径，并观察ROS强度降低后VSMCs死亡方式的变化。对于p53途径，采用p53蛋白印迹、透射电子显微镜、激光共聚焦显微镜进行观察。使用CYP阻断剂和诱导剂观察大黄素作用特点(抑制VSMCs增殖、促进ROS)是否发生变化。利用基因芯片观察上述信号通路基因转录水平的变化。结果：(1)低浓度大黄素(3-1、6.3μg／ml)抑制VSMCs增殖，高浓度大黄素(25.0μg／ml)促进VSMCs死亡。GOG1期细胞呈大黄素浓度依赖性增多，而S期细胞则显著减少，25μg／ml大黄素组S期细胞较正常对照组减少达80％(7.40±1.65％vs．39.05±5.88％，p＜0.05)。不同浓度大黄素均可抑制VSMCs迁移，25μg／ml抑制率为89.6％(p＜0.05)。(2)大黄素以持续稳定的方式促进细胞内ROS生成(r=0.97，p＜0.01)，NADPH氧化酶抑制剂(DPI或PDTC)可以抑制大黄素促ROS效应，而其他抑制剂(CsA、ABT、α-NF)则无抑制效果。细胞内抗氧化能力并无明显变化。大黄素促使VSMCs凋亡和坏死，以凋亡为主。DPI可以显著阻断200μM过氧化氢诱导的VSMCs坏死和凋亡(凋亡：12.6±0.98％vs．4.0±1.00％，p＜0.05，坏死：32.2±2.99％vs．4.87±2.22％，p＜0.05)，也能显著阻断大黄素促VSMCs凋亡(14.55±2.29％vs．8.77±0.83％，p＜0.05)，对坏死并无明显影响(1.18±0.14％vs．1.25±0.28％。p＞0.05)。乳酸脱氢酶活性测定显示大黄素促进VSMCs坏死，可被DPI轻微减轻。(3)大黄素干预后细胞出现非计划性DNA合成(DNA损伤修复标志)。DNA修复基因表达增强。p53蛋白表达也上调(25μg／ml大黄素组较对照组高1.69倍，p＜0.05)。透射电子显微镜显示细胞核中异染色质增多。DPI未影响大黄素的UDS现象。(4)激光共聚焦显示大黄素能迅速渗透进入细胞内部，但在细胞内的分布呈明显的选择性：绝大多数大黄素分布于细胞浆中，仅有少量分布于细胞核内。(5)CYP抑制剂(ABT、α-NF)或者诱导剂(3-MC、β-NF)对大黄素抑制VSMCs增殖和促ROS生成效应均无明显影响。(6)大黄素呈浓度和时间依赖性促进VSMCs老化。我们还观察到大黄素干预后VSMCs自噬的现象。结论：(1)低浓度大黄素抑制VSMCs增殖，高浓度促进VSMCs死亡。(2)ROS途径和p53途径是大黄素发挥效应的两个主要途径，ROS是大黄素促细胞凋亡效应的主要参与者，p53途径主要参与抑制细胞增殖，也涉及细胞凋亡。(3)培养的平滑肌细胞中没有CYP表达，大黄素自身发挥作用，而非代谢产物。第二部分MTT法用于测定大黄素细胞毒性作用的效果背景：MTT法被广泛用于测定药物的细胞毒性作用，但其特异性和敏感性也可以受一些因素的影响，如有颜色物质。我们将探索大黄素自身是否也是干扰因素。方法：血管平滑肌细胞培养于M199培养液中。使用分光光度计测定大黄素和甲(?)的吸光度(O．D．)。结果：在不同溶剂中，大黄素具有不同的吸收光谱。溶剂中存在水分时吸收光谱将向右移，与甲(?)的吸收光谱明显重叠。大黄素呈浓度依赖性直接将MTT还原为甲(?)，大黄素的还原能力可被血清显著抑制。大黄素在细胞内含量非常微弱且其代谢转化非常缓慢。结论：大黄素可以干扰MTT法的准确性，改良后的MTT法可用于评价大黄素的细胞毒性。更多还原

【Abstract】 Part I. The antiproliferative activity of emodin is through ROS-dependent and p53-dependent pathway in human smooth muscle cells Background: Emodin （1, 3, 8-Trihydroxy-6-methylanthraquinone）, a natural anthraquinoid compound, has the remarkably suppressing activity on the various tumor cells proliferation. However, the mechanism of effect of emodin on the smooth muscle cells has remained largely unknown. Emodin can induce apoptotic response in the human hepatocellular carcinoma cell line Mahlavu and human promyeloleukemic HL-60 cells. Preincubation of Mahlavu cells with catalase （CAT） showed partially preventive effect on emodin-induced apoptotic responses. However, preincubation of HL-60 cells with free radical scavenging agents including catalase showed no preventive effect. Whether the antiproliferative activity of emodin is through reactive oxygen species （ROS）-dependent apoptotic pathway is still in argument. Meanwhile, emodin exhibits mutagenicity in the Salmonella typhimurium mutagenicity assay and genotoxicity in the mammalian test systems. DNA damage-inducing protein p53 can inhibit cell growth, by arresting proliferation or inducing apoptosis. So, the activation of p53 may be another pathway participated in the inhibition of proliferation induced by emodin. Emodin can be transformed into at least 10 anthraquinoid metabolites through cytochrome P450 enzyme （CYP）. 2-hydroxyemodin, a main metabolite converted from emodin, is a little more cytotoxic than emodin. The characteristic of antiproliferation of emodin might be influenced by CYP activity which can be enhanced by CYP inducer or suppressed by CYP inhibitor. In the present study, we investigated whether emodin has antiproliferative effect on the smooth muscle cells and whether the effect of emodin is through ROS-dependent and p53-dependent pathway and whether metabolic transformation of emodin is an important step for the effect of emodin. Materials and methods: Vascular smooth muscle cells were cultured in M199 medium supplemented with 15% fetal bovine serum （FBS）. The antiproliferative activity of emodin was observed by several tests, such as total cell count, MTT assay, lactate dehydrogenase （LDH） release, cell cycle analysis, and cell migration. The types of cell death induced by emodin were determined by annexin V-PE and 7-AAD staining. To investigate ROS pathway,celluar ROS was measured. When ROS could be suppressed by inhibitor, the types of cell death were measured again. To investigate p53 pathway, the level of protein p53 was measured by western blot. Transmission electron microscope and laser confocal microscope had also been used. To investigate emodin metabolic pathway, CYP inducer or inhibitor was used to alter CYP enzyme activity. cDNA MicroArray was used to determined which gene expression is altered after emodin treatment.Results: （1） The loss in viability of VSMCs was an emodin dose-dependent manner. Emodin delayed the number of VSMCs entering DNA synthesis （S） phase in a concentration-dependent manner. The percent of S in 25.0 ug/ml emodin group was significantly lower than that in control group （7.40±1.65% vs. 39.05±5.88%, p<0.05）. VSMCs migration was also inhibited by emodin, 89.6% decrease of migrated cell numbers in 25 μg/ml emodin group. （2） ROS generation increased in emodin dose-dependent manner （r=0.97, p<0.01）. The cellular ROS level remained unchanged during a 72 h period. Increased ROS could be inhibited by NADPH oxidase inhibitor DPI or PDTC, but not other inhibitors （CsA, ABT, α-NF）. The change of cellular antioxidantive potential was not significantly. Two types of cell death, apoptosis and necrosis, had been found in VSMCs treated with emodin for 24 hours. When VSMCs was precubated with DPI before emodin added in, the ratio of apoptotic cells significantly decreased from 14.55±2.29% to 8.77±0.83% （p<0.05） and the ratio of necrotic cells didn’t change significantly （1.18±0.14% vs. 1.25±0.28%）. DPI could also significantly inhibit the cell death induced by 200μM H₂O₂ （apoptotic cell: 12.6+0.98% vs. 4.0±1.00%, p<0.05; necrotic cell: 32.2±2.99% vs. 4.87±2.22%, p<0.05）. LDH release can be enhanced by emodin and can be partially inhibited by DPI pretreatment. （3） Unshedueled DNA synthesis, a symbol of DNA damage, was observed after emodin treatment and can not be eliminated by DPI. The level of protein p53 in 25 ug/ml emodin group was 1.69-fold more than that in control group. Heterochromatin was also increased. （4） The intensity of fluorescence of emodin increased rapidly and reached plateau in 15s. However, the distribution of emodin was selective, mostly in cytoplasma and scarcely in nucleus. （5） Neither CYP inducers （ABT, α-NF） nor CYP inhibitors （3-MC, β-NF） had impact on the effects of emodin, such as ROS generation and proliferation inhibition. （6） Emodin accelerated cellsenescence in a dose and time dependent manner. Cell autophagy had also been observed.Conclusions: （1） Emodin can inhibite human VSMCs proliferation and induce cell death （majority as apoptosis and minority as necrosis ）. （2） The effect of emodin is through ROS-dependent and p53-dependent pathway. ROS pathway mainly participated in the apoptotic response and p53 pathway participated both in the aopototic response and in the proliferation inhibition. （3） There is no evidence to support the existence of CYP. Emodin played the role of inhibitor of VSMCs by itself, not its metabolite.Part II. Evaluation of MTT assay for measurement of emodin-inducedcytotoxicity.MTT （3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide） tetrazolium assay is widely used for measuring cytotoxicity induced by emodin. Since the specifity and sensitivity of MTT assay may be influenced by many factors including coloring substances, we therefore investigated whether the absorption spectrum of emodin partially overlapped with that of formazan and the reasons for these influences in the present study. We cultured vascular smooth muscle cells （VSMCs） in M199 medium. The optical density （O.D.） of emodin or formazan was measured by spectrophotometer. We observed that emodin has different absorption spectrum in different solvent. The solvents containing water induced shift of the absorption curve of emodin to right, which increased the overlap of absorption curve of emodin and formazan. The increase of the formazan, which was converted from MTT tetrazolium salt by emodin, was paralleled to the concentration of emodin. The intrinsic reductive potential of emodin can be partially suppressed by serum. Emodin in cells was very tiny and its metabolic transformation was quite slow. These data suggest that emodin can alter the accuracy of MTT assay and that modified MTT assay is valuable in emodin-induced cytotoxicity.更多还原